Chloroquine gel and preparation method and application thereof

ABSTRACT

A chloroquine gel and a preparation method and application thereof. A chloroquine nanosphere includes a water-soluble nanosphere carrier, and chloroquine or a chloroquine derivative, wherein a mass ratio of the chloroquine or the chloroquine derivative to the water-soluble nanosphere carrier is no more than 1:3.

BACKGROUND Technical Field

The present invention belongs to the field of biological medicinetechnologies, and more particularly, relates to a chloroquine gel fortreating external genitalia infection and other cutaneous warts and apreparation method and application thereof.

Description of Related Art

Genital infections, also called sexually transmitted diseases, refer toa group of diseases mainly transmitted by sexual contact. More than 20infectious diseases caused by sex behaviors or similar sex behaviors areincluded in the category of genital infections internationally. Sincethe 1980s, floating population has been increasing day by day. With thechange of sexual concept, unsafe sex behaviors have increased.Reproductive tract infection and sexually transmitted infection areincreasing in China, and have spread from the coast to the inland anddeveloped from the city to the countryside, so that the situation isquite severe. According to statistics, there are about 400,000 AIDSpatients in China today, HIV infection is about 1 in 1,000, and abouthundreds of people are infected every day, with characteristics of alarge sexual transmission ratio and the transmission from a high-riskgroup to a general group. According to statistics, the number ofpatients suffering from external genitalia infection of both sexes inChina is still increasing year by year in recent years, an incidencerate in many areas ranks second and third in an order of infectiousdiseases, an incidence rate of lower genital tract infection amongmarried women is as high as 50%, and 70% of women at least suffered fromvulvovaginal candidiasis once in their lifetime, 5% to 10% of whichsuffered from recurrent vaginal candidiasis and 90% of which wereinfected with human papillomavirus. An incidence rate of the externalgenitalia infection of both sexes is increasing so rapidly that adifficulty in control has become a serious social and public healthproblem to the world. Therefore, effective prevention and treatment ofthe external genitalia infection of both sexes have become very seriousand urgent tasks at present.

The external genitalia infection is mainly treated by aiming at apathogen in modern medicine. In terms of prevention and treatment, thereare many kinds of pathogenic microorganisms causing the reproductivetract infection, and the variation and drug resistance of the pathogenmake it extremely difficult to select an antibiotic medicine to preventthe reproductive tract infection. At present, in clinical practice, thetreatment of the external genitalia infection is mostly based onantibiotic or disinfectant administration. However, since irritation ofa disinfectant to a local mucosa destroys an environment in a vagina,the use of the disinfectant is limited, and the disinfectant is usuallyonly suitable for local treatment. For bacterial genital infection, anantibiotic therapy often brings drug resistance and side effects.Therefore, it is of great significance to develop a new and highlyeffective medicine for external use with antibacterial and antiviraleffects for the effective prevention and treatment of the externalgenitalia infection.

Chloroquine has always been used as a medicine to prevent and treatplasmodium infection, and is often used in gene transfection experimentsto improve a transfection efficiency. The high-concentration chloroquineis found to have an effect of inhibiting and killing cryptococcus invitro through in-vitro studies, and effects of the chloroquine on twovarieties of the cryptococcus are not different; and meanwhile, thechloroquine can enhance an anti-cryptococcal ability of amphotericin Band has a synergistic antibacterial effect with the amphotericin B. Atpresent, many studies focus on an anti-HIV activity of the chloroquine.Studies have shown that the chloroquine has a broad-spectrum anti-HIVactivity, and the chloroquine not only is aimed at a laboratory strain Bevolved by HIV-1, but also has a great effect on primary isolates A, B,C, D and E evolved by HIV-1. It seems that a main mechanism of ananti-HIV effect of the chloroquine is to inhibit glycosylation of agp120 virus envelope protein, resulting in that a newly generated virusparticle has a severely reduced infectivity. Meanwhile, it is believedin studies that the chloroquine is an aminoquinine membrane-penetratingagent, which can be embedded into a double-stranded DNA and change adouble helix form of a DNA by unwinding. It is believed inbacteriological studies that the chloroquine can be used as a kind ofintercalators such as acridine orange and ethidium bromide, which canselectively inhibit a covalent bond of a small plasmid DNA, and bychanging a density of DNA supercoils, the closure of the supercoil canbe changed, thus inhibiting the proliferation of bacteria andvirus-infected cells. A DNA of a human papillomavirus (HPV) causing acervical cancer is such a plasmid, which is a supercoiled complex DNA,and performs DNA replication and RNA transcription. In turn, thechloroquine has been proved to be able to inhibit the plasmid DNA andinterfere with an activity of DNA polymerase by changing a superhelixdensity of the plasmid DNA. Due to the alkalescence, the chloroquine hasbeen proved to cause mitochondrial dysfunction, such as accumulation ofendosomes/lysosomes, change of a pH value and possible apoptosis. Inaddition, the chloroquine has been proved to reactivate inactivated P53(HPV degradation/P53 inactivation), be connected to melanin (productionof melanin occurs on the same layer of skin as HPV replication: a basallayer of epidermis), and prevent release of iron ions (ironcatalyst/energy dependence of HPV during replication). Iron is necessaryfor DNA replication, and the iron also acts as an electronic catalyst intransportation and storage of oxygen. However, the iron is also a vitalsubstance for pathogen survival and virus replication. Inhibition ofiron particles is also an important mechanism for the chloroquine tokill viruses and virus-infected cells.

At present, there's no medicine of chloroquine for external use,especially the medicine that has no external preparation with lessirritation and wider curative effect. In clinical application, thechloroquine is mainly used to treat an acute malaria attack and controla malaria symptom. The chloroquine can also be used for treating hepaticamebiasis, clonorchiasis, paragonimiasis, connective tissue disease,etc. The chloroquine can also be used for treating photosensitivediseases such as erythema solare disease. A tablet and an injection areused for treating malaria with large dosage and long course oftreatment, which may have serious side effects on gastrointestinal tractand skin. In addition, the chloroquine has certain irritation on skin,causing skin damage and various types of rashes. Therefore, how toreduce the irritation of the chloroquine and the side effects, andimprove an application value of the chloroquine by changing a dosageform has become the main problem at present.

A medicine microsphere refers to a tiny spherical entity formed bymedicine dispersion or adsorption in a high-molecular polymer matrix. Amicrosphere preparation has a long-acting, sustained-release or targetedeffect, which can greatly improve convenience and compliance of patientsin medication, with outstanding advantages in clinic, so that thepreparation is a potential dosage form. In addition, a microspherepreparation product has a great added value and a broad market prospect,and has become a hot spot in medicine research and development in recentyears.

SUMMARY

An objective of the present invention is to overcome the defects in theprior art above, and provide a chloroquine nanosphere, which selects anatural polymer compound as a microsphere carrier, has a simplepreparation process and a small side effect of a product, gives fullplay to a synergistic effect of chloroquine and the microsphere carrier,has obvious anti-inflammatory, antibacterial and antiviral effects,reduces irritation of the chloroquine to skin, promotes wound healing,can control a release speed of a medicine, and overcomes defects of aweak antibacterial effect of the existing microsphere carrier and largeirritation of the chloroquine.

Another objective of the present invention is to provide a preparationmethod for the chloroquine nanosphere applied to external genitalia andother cutaneous warts.

One another objective of the present invention is to provide a newapplication for the chloroquine nanosphere above.

Still another objective of the present invention is to provide a productfor preventing and treating external genitalia infection and/or othercutaneous warts.

The objectives of the present invention above are achieved through thefollowing technical solutions.

A chloroquine nanosphere includes a water-soluble nanosphere carrier,and chloroquine or a chloroquine derivative.

The inventor unexpectedly discovers that the nanosphere which isprepared by combining the water-soluble nanosphere carrier with thechloroquine or the chloroquine derivative, has a long action time, amucosal adhesiveness, a local retention and a self-degradability,reduces irritation of the chloroquine, has no irritation on the externalgenitalia, overcomes defects of a large side effect and inconvenient usein an existing external genitalia infection treatment method at the sametime, and can effectively solve a side effect and a medicine resistanceof the chloroquine; the nanosphere of the present invention caneffectively prevent or treat external genitalia infection and othercutaneous wart diseases, such as vaginitis, flat warts and the like, andeven has a remarkable synergistic effect; and not only an applicationrange of the chloroquine is expanded, but also a phenomenon of medicineresistance caused by abuse of existing antibacterial medicines isreduced.

The present invention further relates to a preparation method for thechloroquine nanosphere above, which includes the following steps:

S1: after dissolving the water-soluble nanosphere carrier, adding thechloroquine or the chloroquine derivative, and evenly mixing andstirring to form an aqueous phase, wherein a mass ratio of thechloroquine or the chloroquine derivative to the water-solublenanosphere carrier is no more than 1/3;

S2: adding an emulsifier into an oil phase matrix to form an oil phase;adding the aqueous phase into the oil phase, wherein a volume ratio ofthe aqueous phase to the oil phase is 1:1 to 1:6; and emulsifying at10000-20000 r/min for 10-30 minutes to obtain a nanosphere emulsion; and

S3: adding a precipitant into the nanosphere emulsion, after evenlymixing, standing for 2-8 minutes, then dehydrating, performinghigh-speed centrifugation and cleaning, and drying to obtain thechloroquine nanosphere.

Preferably, a mass ratio of the chloroquine or the chloroquinederivative to the water-soluble nanosphere carrier ranges from 1:3 to1:5.

More preferably, the mass ratio of the chloroquine or the chloroquinederivative to the water-soluble nanosphere carrier is 1:3.

Preferably, the chloroquine derivative is selected from one or more ofhydroxychloroquine, chloroquine phosphate or chloroquine sulfate.

Preferably, a loading rate of the chloroquine or the chloroquinederivative ranges from 3.0% to 21.6%.

More preferably, the loading rate of the chloroquine or the chloroquinederivative is 12.5%.

Preferably, the water-soluble nanosphere carrier is selected from one ormore of water-soluble chitosan, water-soluble carrageenan orwater-soluble starch.

Preferably, the water-soluble nanosphere carrier is the water-solublechitosan.

More preferably, a deacetylation degree of the water-soluble chitosanranges from 80% to 95%, and a viscosity-average molecular weight thereofranges from 3000 to 5000.

Preferably, the volume ratio of the aqueous phase to the oil phase inthe step S2 is 1:3.

Preferably, the high-speed centrifugation in the step S3 is performed at1500-2000 r/min for 5-10 minutes.

More preferably, the high-speed centrifugation in the step S3 isperformed at 1500 r/min for 10 minutes.

Preferably, in the step S3, the precipitant is added into the nanosphereemulsion at a speed of 1 to 5 drops per second.

Preferably, a volume ratio of the emulsifier to the precipitant is(1-1.5):(30-50).

More preferably, the volume ratio of the emulsifier to the precipitantis 1:50.

Preferably, the oil phase matrix is vegetable oil; and the precipitantis an alkaline organic phase.

More preferably, the alkaline organic phase is a sodiumhydroxide-n-propanol mixed solution.

More preferably, a pH value of the alkaline organic phase ranges from8.5 to 10.0.

More preferably, the pH value of the alkaline organic phase is 9.0.

Preferably, the vegetable oil is selected from one or more of corn oil,olive oil, peanut oil, soybean oil, rapeseed oil and other vegetableoils.

Preferably, the emulsifier is selected from one or more of Tween-20,Tween-80 and Span 80.

Application of the chloroquine nanosphere above in preparing anantibacterial and/or antiviral product, and application of thechloroquine nanosphere above in preparing a product for preventing andtreating external genitalia infection and/or other cutaneous warts arealso included in the scope of protection of the present invention.

The product including the chloroquine nanosphere above can obviouslyreduce skin irritation of the chloroquine, promote wound healing, play aslow release effect, and improve a biological activity at the same time,and is applied to treat external genitalia infection and/or othercutaneous warts.

Preferably, the external genitalia infection includes viral vaginitis,condyloma acuminatum, bacterial vaginosis, fungal-infectious vaginitisor trichomonal vaginitis.

Preferably, the other cutaneous warts include flat warts and warts atnon-genital parts caused by HPV infection.

The chloroquine nanosphere has good therapeutic effects on the externalgenitalia infection including viral vaginitis, condyloma acuminatum,bacterial vaginosis, fungal-infectious vaginitis or trichomonalvaginitis, and the other cutaneous warts including flat warts and wartsat non-genital parts caused by HPV infection.

The present invention further provides a product for preventing andtreating external genitalia infection and/or other cutaneous warts,which includes chloroquine or a chloroquine derivative, and furtherincludes one or more of water-soluble chitosan, water-solublecarrageenan or water-soluble starch.

Preferably, a mass ratio of the chloroquine or the chloroquinederivative to the water-soluble chitosan, the water-soluble carrageenanor the water-soluble starch is not more than 1/3.

More preferably, the mass ratio of the chloroquine or the chloroquinederivative to the water-soluble chitosan, the water-soluble carrageenanor the water-soluble starch ranges from 1:3 to 1:5.

More preferably, the mass ratio of the chloroquine or the chloroquinederivative to the water-soluble chitosan, the water-soluble carrageenanor the water-soluble starch is 1:3.

Further, in a preferred embodiment of the present invention, the productfor preventing and treating external genitalia infection and/or othercutaneous warts includes the chloroquine nanosphere above.

More preferably, the chloroquine nanosphere accounts for 1% to 6% of atotal weight of the product.

Further, in a preferred embodiment of the present invention, the productis gel, ointment, cream, effervescent tablet, vaginal tablet, capsule,film agent or suppository.

The product of the present invention can be processed into a semi-solidpreparation such as gel, cream and the like, or a solid preparation suchas tablet, effervescent tablet and the like; and the product of thepresent invention can obviously improve bitter taste and skin irritationof the chloroquine, promote wound healing, play a slow release effectand improve a biological activity at the same time.

Further, in a preferred embodiment of the present invention, the productis gel.

Compared with the prior art, the present invention has the followingbeneficial effects.

(1) According to the present invention, the water-soluble nanospherecarrier is combined with the chloroquine to prepare an externalpreparation with sterilization and antivirus functions at the same time,which plays an obvious synergistic effect, has certain cleaning andkilling effects on various bacteria and viruses, and cleaning andnursing effects on external genital organs for male and female, reducesinflammatory reactions of the external genitalia, can well improve skinirritation caused by the chloroquine, has no irritation on vaginalmucosa and penis, and has mucosal adhesiveness, local retention andself-degradability, and the preparation not only can prevent bacterialand viral infection, but also can promote micro wound healing, and canbe used for effective treatment of external genitalia infection andwarts, especially health care of external genitalia before and aftersexual life, with an obvious effect, and a cool and comfortable effect.

(2) The present invention mainly uses the water-soluble nanospherecarrier combined with the synergist chloroquine as a main component. Theprescription is stable, and a medicine exists in a form of microsphereso that a detention time of the medicine at a treated part can beprolonged, enabling an action time to be longer, and improving abioavailability and a therapeutic effect of the medicine. The medicineis simple and convenient in preparation method, easy in synthesiswithout toxic and side effects, and has high safety; moreover,industrial application of a new medicine can be performed, thusproviding a new direction for development and application of anantibacterial-active and antiviral-active medicine.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a chloroquine-chitosan nanosphere.

FIG. 2 is a standard curve for high-performance liquid-phase detectionof chloroquine.

FIG. 3 shows a content of chloroquine in the chloroquine-chitosanmicrosphere in high-performance liquid-phase detection.

FIG. 4 shows drug release of the chloroquine-chitosan nanosphere and thechloroquine microsphere.

FIG. 5 shows gel products with different ratios observed on a smoothglass plane (a mass ratio of chloroquine phosphate to chitosan isrespectively 1:5, 1:3, 1:1, 1:0.5 from No. 1 to No. 4 gel products,wherein No. 5 is a gel product of pure chitosan).

FIG. 6 shows flowing of the gel products with different ratios on thesmooth glass plane in 1 minute (the mass ratio of the chloroquinephosphate to the chitosan is respectively 1:5, 1:3, 1:1, 1:0.5 from No.1 to No. 4 gel products, wherein No. 5 is the gel product of purechitosan).

FIG. 7 shows H-E staining results of an irritation test on damaged skin,wherein Group A is a control group with a blank gel matrix, including A1of FIG. 7 (tested substance side) and A2 of FIG. 7 (negative controlside); Group B is a chloroquine-chitosan nanosphere gel group 1 with amass ratio of chloroquine phosphate to chitosan being 1:3, including B1of FIG. 7 (tested substance side) and B2 of FIG. 7 (negative controlside); Group C is a chloroquine-chitosan nanosphere gel group 2 with amass ratio of chloroquine phosphate to chitosan being 1:5, including C1of FIG. 7 (tested substance side) and C2 of FIG. 7 (negative controlside); Group D is a chloroquine-chitosan nanosphere gel group 3 with amass ratio of chloroquine phosphate to chitosan being 1:0.5, includingD1 of FIG. 7 (tested substance side) and D2 of FIG. 7 (negative controlside); and Group E is a chloroquine phosphate gel group, including E1 ofFIG. 7 (tested substance side) and E2 of FIG. 7 (negative control side).

FIG. 8 shows H-E staining results of an animal test on herpes virusvaginitis, wherein A of FIG. 8 is a blank control group; B of FIG. 8 isa model control group; C of FIG. 8 is a chloroquine-chitosan nanospheregel group 1 with a mass ratio of chloroquine phosphate to chitosan being1:3; and D of FIG. 8 is a chloroquine-chitosan nanosphere gel group 2with a mass ratio of chloroquine phosphate to chitosan being 1:0.5; andE of FIG. 8 is a chloroquine phosphate gel group.

FIG. 9 shows H-E staining results of an animal test on HPV condylomaacuminatum, wherein A of FIG. 9 is a model control group; B of FIG. 9 isa chloroquine-chitosan nanosphere gel group 1 with a mass ratio ofchloroquine phosphate to chitosan being 1:3; C of FIG. 9 is achloroquine chitosan nanosphere gel group 2 with a mass ratio ofchloroquine phosphate to chitosan being 1:0.5; and D of FIG. 9 is achloroquine phosphate gel group.

DESCRIPTION OF THE EMBODIMENTS

The invention is further described hereinafter with reference to thespecific embodiments, but the embodiments are not intended to limit theinvention in any form. Any simple modifications or substitutions made tothe methods, steps or conditions of the invention without departing fromthe spirit and essence of the invention shall all fall within the scopeof the present invention. Unless otherwise specified, the technicalmeans used in the embodiments are conventional means well known to thoseskilled in the art.

The reagents and materials used in the following embodiments arecommercially available unless otherwise stated.

Embodiment 1 Preparation of Chloroquine-Chitosan Nanosphere

1. Preparation Method

In S1, 3 g of water-soluble chitosan was dissolved into 100 mL of water,added with 1.0 g of chloroquine phosphate, and evenly mixed and stirredto form an aqueous phase, wherein a degree of deacetylation of thewater-soluble chitosan was 80%, and a viscosity-average molecular weightthereof was 5000.

In S2, 300 mL of corn oil was used as an oil phase matrix, and 1 mL ofan emulsifier Tween-20 was added into the oil phase matrix to form anoil phase; and the aqueous phase was added into the oil phase, wherein avolume ratio of the aqueous phase to the oil phase was 1:3, and thenemulsification was performed at 15000 r/min for 30 minutes to obtain ananosphere emulsion.

In S3, a sodium hydroxide-n-propanol mixed solution was used as aprecipitant, 10 mL of n-propanol was firstly added with several drops ofsaturated sodium hydroxide solution to adjust a pH value to 9, then themixed solution was evenly mixed and added into the nanosphere emulsionat a speed of 1 to 5 drops per second, a volume ratio of the emulsifierto the precipitant was controlled to be 1:50, then a mixture obtainedwas stood for 5 minutes, and added with a proper amount of n-propanolfor dehydration; and then the mixture was centrifuged at 1500 r/min for10 minutes, cleaned, and dried, thus obtaining the chloroquine-chitosannanosphere.

2. Product Characterization

(1) The prepared chloroquine-chitosan nanosphere is shown in FIG. 1,wherein a particle diameter of the prepared chloroquine-chitosannanosphere ranges from 100 nm to 800 nm.

(2) As shown in FIG. 2 and FIG. 3, a loading rate of the chloroquine inthe chloroquine-chitosan microsphere is 12.5% via determination by highperformance liquid chromatography.

Embodiment 2 Preparation of Chloroquine-Chitosan Nanosphere

1. Preparation Method

In S1, water-soluble chitosan was dissolved in water, then added withhydroxychloroquine, and evenly mixed and stirred to form an aqueousphase, and a mass ratio of the hydroxychloroquine to the water-solublechitosan was controlled to be 1:5, wherein a degree of deacetylation ofthe water-soluble chitosan was 90%, and a viscosity-average molecularweight thereof was 3000.

In S2, 300 mL of corn oil was used as an oil phase matrix, and 1 mL ofan emulsifier Tween-20 was added into the oil phase matrix to form anoil phase; and the aqueous phase was added into the oil phase, wherein avolume ratio of the aqueous phase to the oil phase was 1:1, and thenemulsification was performed at 10000 r/min for 30 minutes to obtain ananosphere emulsion.

In S3, a sodium hydroxide-n-propanol mixed solution was used as aprecipitant, 10 mL of n-propanol was added with several drops ofsaturated sodium hydroxide solution to adjust a pH value to 8.5, thenthe mixed solution was evenly mixed and added into the nanosphereemulsion slowly, a volume ratio of the emulsifier to the precipitant wascontrolled to be 1.5:50, then a mixture obtained was stood for 2minutes, and added with a proper amount of n-propanol for dehydration;and then the mixture was centrifuged at 2000 r/min for 5 minutes,cleaned, and dried, thus obtaining the chloroquine-carrageenannanosphere, with a particle diameter ranging from 200 nm to 800 nm, anda loading rate of chloroquine of 10.3%.

Embodiment 3 Preparation of Chloroquine-Chitosan Nanosphere

1. Preparation Method

In S1, water-soluble chitosan was dissolved in water, then added withchloroquine sulfate, and evenly mixed and stirred to form an aqueousphase, and a mass ratio of the chloroquine sulfate to the water-solublechitosan was controlled to be 1:7, wherein a degree of deacetylation ofthe water-soluble chitosan was 90%, and a viscosity-average molecularweight thereof was 4000.

In S2, olive oil was used as an oil phase matrix, and an emulsifierTween-80 was added into the oil phase matrix to form an oil phase; andthe aqueous phase was added into the oil phase, wherein a volume ratioof the aqueous phase to the oil phase was 1:6, and then emulsificationwas performed at 20000 r/min for 10 minutes to obtain a nanosphereemulsion.

In S3, a sodium hydroxide-n-propanol mixed solution was used as aprecipitant, n-propanol was firstly added with several drops ofsaturated sodium hydroxide solution to adjust a pH value to 10, then themixed solution was evenly mixed and added into the nanosphere emulsionslowly, a volume ratio of the emulsifier to the precipitant wascontrolled to be 1.5:30, then a mixture obtained was stood for 8minutes, and added with a proper amount of n-propanol for dehydration;and then the mixture was centrifuged at 1500 r/min for 10 minutes,cleaned, and dried, thus obtaining the chloroquine-starch nanosphere,with an average particle diameter of 400 nm, and a loading rate ofchloroquine of 8.6%.

Embodiment 4 Preparation of Chloroquine-Chitosan Nanosphere Gel

1. Prescription of gel:

Chloroquine-chitosan 1% nanosphere Carboxymethyl cellulose 2%Ethylparaben 0.2%   Glycerol 30%  Peppermint oil 0.1%   Purified waterSupplemented to 100%

2. The preparation method included the following steps.

(1) Preparation of microsphere: the microsphere was prepared accordingto the preparation steps of the chloroquine-chitosan nanosphere in theEmbodiment 1.

(2) 20 g of carboxymethyl cellulose was added with 300 g of glycerol(prescription dosage) and 2 g of ethylparaben, then a mixture obtainedwas evenly stirred, and fully swelled, and used as a gel matrix; 10 g ofthe chloroquine-chitosan nanospheres were dissolved with 100 mL ofwater, added into the gel matrix in batches and stirred. 1 ml ofpeppermint oil was added, and water was added to 1000 mL to prepare anantibacterial and antiviral chloroquine-chitosan nanosphere gel suitablefor external genitalia.

3. Pure chloroquine gel was prepared by the same method, and releaserates of chloroquine in the chloroquine-chitosan nanosphere gel and thechloroquine gel were compared. As shown in FIG. 4, thechloroquine-chitosan nanosphere gel could slowly and stably release thechloroquine. The chloroquine-chitosan nanosphere gel prepared by thepresent invention has no sudden release phenomenon at the early stage,and a release speed of the chloroquine-chitosan nanosphere gel is almostnot affected by a medium. The chloroquine-chitosan nanosphere gelpresents a good slow release effect, which is beneficial for sustainedrelease of a medicine after external administration.

Embodiment 5 Preparation of Chloroquine-Chitosan Nanosphere Gel

1. Prescription of gel:

Chloroquine-chitosan   5% nanosphere Carboxymethyl cellulose 0.5%Ethylparaben 0.5% Glycerol  30% Peppermint oil 0.1% Purified waterSupplemented to 100%

2. The preparation method included the following steps.

(1) Preparation of microsphere: the microsphere was prepared accordingto the preparation steps of the chloroquine-chitosan nanosphere in theEmbodiment 1.

(2) Carboxymethyl cellulose was added with glycerol (prescriptiondosage) and 800 mL of water, then a mixture obtained was evenly stirred,and fully swelled, and used as a gel matrix; the chloroquine-chitosannanospheres were dissolved with 100 mL of water, added into the gelmatrix in batches and stirred. Ethylparaben was dissolved with 50 mL ofhot water at 80° C. to 85° C., and added into the gel matrix when atemperature was reduced below 40° C., and stirred evenly. Peppermint oilwas added, and water was added to 1000 mL to prepare an antibacterialand antiviral chloroquine-chitosan nanosphere gel suitable for externalgenitalia.

3. According to the steps above, with remaining process conditionsunchanged, mass ratios of chloroquine phosphate to chitosan were changedto prepare gels 1, 2, 3 and 4 respectively; and meanwhile, according tothe preparation steps of the embodiment above, with remaining processconditions unchanged, pure chitosan was used as a main active componentto prepare gel 5, without adding with chloroquine or a chloroquinederivative, as shown in the following table.

TABLE 1 Results of Preparation of Nanosphere Gel with Different Ratiosof Chloroquine to Chitosan Mass ratio of chloroquine Effective phosphateto pH Viscosity chloroquine chitosan Character value (mp/s) content (%)Gel 1 1:5 Pale yellow 4.27 11868 0.84 semitransparent gel with eventexture and faint mint fragrance, which can flow downwardly in a columnGel 2 1:3 Beige non- 4.08 9246 1.67 transparent gel with even textureand faint mint fragrance, which can flow downwardly in a column Gel 31:1 Beige non- 3.97 6217 2.50 transparent paste with faint mintfragrance and small particles Gel 4  1:0.5 Earthy yellow 3.77 5890 5.03non-transparent paste with faint mint fragrance and large particles Gel5 Pure Orange 4.79 20969 0 chitosan transparent gel with faint mintfragrance and even texture

FIG. 5 is a shape diagram of gel products with different ratios observedon a smooth glass plane (a mass ratio of chloroquine phosphate tochitosan is respectively 1:5, 1:3, 1:1, 1:0.5 from No. 1 to No. 4 gelproducts, wherein No. 5 is a product of pure chitosan); and FIG. 6 showsflowing of the gel products with different ratios on the smooth glassplane in 1 minute.

The experiment showed that when the ratio of the chloroquine phosphateto the chitosan was 1:1 or 1:0.5, the gel prepared was uneven intexture, had small particles, and had a poor fluidity. When the ratio ofthe chloroquine phosphate to the chitosan was 1:5 or 1:3, the productwith gel of even texture, a pH suitable for vaginal and skinadministration, a moderate viscosity, color and odor meeting the needsof different populations, and a stable chloroquine content was prepared.However, when the mass ratio of the chloroquine phosphate to thechitosan was 1:5, the chloroquine content was lower and a medicineeffect was limited to a certain extent, and under comprehensiveconsideration, when the mass ratio of the chloroquine phosphate to thechitosan was 1:3, the product was better in performance.

Embodiment 6 Irritation Test of Chloroquine-Chitosan Nanosphere Gel onDamaged Skin

1. Experimental Method

(1) New Zealand rabbits were used as experimental objects, left andright skins of the same body were compared with each other, a“Tic-Tac-Toe” symbol (with an area of about 3.0 cm×3.0 cm) was marked onskins of administration parts on left and right sides respectively byusing an intra-individual left/right skin self-comparison method. 1.0 gof tested substance was given to the left side, wherein anadministration dose was 9 cm²/per rabbit, an application area of eachtime was 3.0 cm×3.0 cm. 1.0 g of white vaseline was given to the rightside as a negative control side. Animals in each group were administeredonce a day for 28 consecutive days.

Groups of the tested substances were respectively as follows:

Group A: a control group, with a blank gel matrix,

Group B: a chloroquine-chitosan nanosphere gel group 1, wherein a massratio of chloroquine phosphate to chitosan was 1:3,

Group C: a chloroquine-chitosan nanosphere gel group 2, wherein a massratio of chloroquine phosphate to chitosan was 1:5,

Group D: a chloroquine-chitosan nanosphere gel group 3, wherein a massratio of chloroquine phosphate to chitosan was 1:0.5, and

Group E: a chloroquine phosphate gel group.

Before each application, and 1 hour after each medicine removal, and 1hour, 24 hours, 48 hours, 72 hours and 14 days after the last medicineremoval on the 28^(th) day, erythema and edema at the administrationparts were observed and recorded respectively on each observation timepoint for the animals in each group above, and the erythema and theedema were scored. Meanwhile, it was necessary to observe whetherpigmentation, bleeding point, skin roughness or skin thinning occurredat the administration parts, and to record occurrence and regressiontime of the pigmentation, the bleeding point, the skin roughness or theskin thinning.

(2) For the animals in the five groups above, left and right skins ofthe administration parts of the animals in each group were respectivelysubjected to histopathological examination on the 14^(th) day after thelast medicine removal.

2. Results

(1) Skin Observation Results of Each Group

Test Results:

On the negative control side (right side), the skins of theadministration parts of the animals in each tested substance group hadno obvious irritation reaction on each observation time point, so thatit could be judged that a result of a negative control substance (whitevaseline) was negative.

Group A: no obvious irritant reaction was found on all the observationtime points, so that it could be judged that the blank gel matrix had noirritation on skin.

Group B: the skins of the administration parts on the tested substanceside (left side) of 6/6 animals had slight erythema or edema on thevisual observation time point from the 1^(st) day to the 12^(th) day, noobvious abnormality was found in observation on the administration partsof each animal from the 12^(th) day to the time before necropsy, andhistopathological examination results of 72 hours after and the 14^(th)day after the last medicine removal showed that the skins of theadministration parts on the tested substance side of 6/6 animals hadslight edema in 2 cases and no obvious abnormal change in other cases.Therefore, the irritation above tended to be judged in that the medicineslightly aggravated a mechanical stimulation after the skin was damaged.

Group C: the skins of the administration parts on the tested substanceside (left side) of 6/6 animals had slight erythema or edema on a visualobservation time point from the 1^(st) day to the 12^(th) day, noobvious abnormality was found in observation on the administration partsof each animal from the 12^(th) day to the time before necropsy, andhistopathological examination results of 72 hours after and the 14^(th)day after the last medicine removal showed that the skins of theadministration parts on the tested substance side of 6/6 animals had noobvious abnormal change. Therefore, the irritation above tended to bejudged as a mechanical stimulation after the skin was damaged.

Group D: 4/6 of the administration parts on the tested substance side(left side) of the 6/6 animals had relatively obvious erythema or edemaon the 12^(th) day, scabs fell off during administration, the skinsurfaces failed to completely heal when the scabs fell off, and woundshealed obviously 72 hours after the last medicine removal.

Group E: 4/6 of the damaged parts of the administration parts on thetested substance side (left side) of the 6/6 animals cracked on the11^(th) day, and tended to be outwards extended, the erythema or theedema at the administration parts was more obvious than that in theprevious period, scabs were continuously regenerated and then fell off,and the skin surface failed to completely heal when the scabs fell off,which was significantly different from the negative control side (theintra-individual right side). Combined with the histopathologicalexamination results of 72 hours after and the 14^(th) day after the lastmedicine removal, the 6/6 animals had obvious abnormalities in the skinsof the administration parts on the tested substance side, wherein 3cases autopsied 72 hours after the last medicine removal showed acertain degree of damage, obvious thickening of an epidermal layer,disorder of arrangement of subepidermal connective tissues,proliferation of fibrous tissues, infiltration of inflammatory cells,scab of epidermis, depression of a part of epidermis, fuzzy structure,etc., and 3 cases autopsied on the 14^(th) day after the last medicineremoval showed local epidermal thickening, disorder of arrangement ofsubepidermal connective tissues, proliferation of fibrous tissues andinfiltration of inflammatory cells.

It could be seen from the experimental results above that irritation ofchloroquine phosphate chitosan nanosphere gel on the damaged skins wassignificantly lower than that of chloroquine phosphate gel (generalexternal preparation) after multiple administration to the damaged skinsof the New Zealand rabbits. For the chloroquine phosphate chitosannanosphere gel, the lower the ratio of the chloroquine/nanospherecarrier was, the smaller the irritation reaction on the skin was, andthe better the repair of the damaged skin after stopping administrationwas. However, the chloroquine gel could cause worse damage on thedamaged skins, when the negative control side was completely repaired,the skins on the chloroquine gel side could not be repaired normally,and the skins on the chloroquine gel side could only recover to acertain extent 14 days after stopping administration, with an obviousdifference, which indicated that the chloroquine had an irritationeffect on the damaged skins, and the chloroquine-chitosan microspherecould significantly reduce the irritation effect, which might be relatedto antibacterial, anti-inflammatory and wound healing promoting effectsof the chitosan itself.

(2) Pathological Examination Results

Group A: as shown in A1 (blank matrix side) and A2 (negative controlside) in FIG. 7, skin epidermis, dermis and accessories were in goodcondition structurally, and inflammatory cell infiltration and smallvessel dilatation were not found.

Group B: as shown in B1 (chloroquine-chitosan nanosphere gel) and B2(negative control side) in FIG. 7, B2 of FIG. 7 showed that the skinepidermis, the dermis and the accessories were in good conditionstructurally, and the inflammatory cell infiltration and the smallvessel dilatation were not found. Compared with the negative controlside, B1 of FIG. 7 showed that a small focal epidermal layer of theskins in the group was slightly thickened, with infiltration of a smallnumber of inflammatory cells.

Group C: as shown in C1 (chloroquine-chitosan nanosphere gel side) andC2 (negative control side) in FIG. 7, C2 of FIG. 7 showed that the skinepidermis, the dermis and the accessories were in good conditionstructurally, and the inflammatory cell infiltration and the smallvessel dilatation were not found. C1 of FIG. 7 showed that a localepidermal layer of the skins in the group was slightly thickened, anumber of cell layers was obviously increased, and the cells grewextendedly to a dermis layer; and arrangement of subepidermal connectivetissues was disordered, with infiltration of some inflammatory cells.

Group D: as shown in D1 (chloroquine-chitosan nanosphere gel side) andD2 (negative control side) in FIG. 7, D2 of FIG. 7 showed that the skinepidermis, the dermis and the accessories were in good conditionstructurally, and the inflammatory cell infiltration and the smallvessel dilatation were not found. Compared with the negative controlside, D1 of FIG. 7 showed that the local epidermal layer in the groupwas slightly thickened, and the number of the cell layers was increased,with infiltration of a small number of inflammatory cells.

Group E: as shown in E1 (chloroquine gel side) and E2 (negative controlside) in FIG. 7, on the negative control side, the skin epidermis, thedermis and the accessories were in good condition structurally, theinflammatory cell infiltration and the small vessel dilatation were notfound, while a local epidermal layer of the skins on the chloroquine gelside had scabs, and the epidermis was obviously thickened; a part ofscab skins were closely connected with a subcutaneous tissue, theepidermal layer was sunken, a structure was fuzzy, and a large number ofinflammatory cells were infiltrated with fibrous tissue hyperplasia; anumber of hair follicles in a dermal layer was decreased significantly;and however, no obvious abnormality was found in a muscular layer andthe subcutaneous tissue.

The pathological examination results above showed that compared with thechloroquine gel, gel of the chloroquine-chitosan microsphere group hadobvious advantages in irritation and healing after medicine withdrawal.In each chloroquine-chitosan microsphere group, the lower the ratio ofthe chloroquine to the nanosphere carrier was, the smaller theirritation reaction to the skin was.

According to the skin observation results and the pathologicalexamination results, the irritation reaction of the chloroquine-chitosannanosphere gel on the skins was obviously smaller than that of thechloroquine gel, which indicated that the chloroquine-chitosannanosphere greatly reduced an irritation side effect of the chloroquineon the skins and promoted wound healing to a certain extent. When a massratio of the chloroquine to the chitosan was not more than 1:3, anirritation effect was smaller and a medicine content was the highest.

Embodiment 7 Animal Test on Herpes Virus Vaginitis

1. Experimental Method

The chloroquine-chitosan nanosphere obtained in the Embodiment 2 wasmade into gel for the animal test on herpes virus vaginitis.

The specific experimental method was that: a number of Hartley guineapigs qualified for adaptive observation were selected, 10 animals wererandomly selected as a blank control group (A), and the other animalswere all infected with herpes virus through vagina for modeling. Aftermodeling, the animals were randomly divided into five groups, and theoverall grouping was as follows:

Group A: a blank control group,

Group B: a model group,

Group C: a chloroquine-chitosan nanosphere gel group 1, wherein a massratio of chloroquine phosphate to chitosan was 1:3,

Group D: a chloroquine-chitosan nanosphere gel group 2, wherein a massratio of chloroquine phosphate to chitosan was 1:0.5, and

Group E: a chloroquine phosphate gel group.

10 animals in each group were administrated vaginally. The blank controlgroup and the model control group were given 0.9% sodium chlorideinjection, while the Groups C, D and E were given corresponding testedmedicine once a day for two weeks. 14 days after administration, avaginal mucosa tissue was taken for fixing and embedding the next day,paraffin wax was continuously cut into sections of 4 μm, and afterhematoxylin-eosin (HE) staining, a pathological condition of the vaginalmucosa tissue was observed under a microscope.

2. Results

(1) Pathological Examination

Group A: results of the blank control group are shown in A in FIG. 8,wherein a vaginal mucosa epithelial cell of the blank control group hada complete structure, and a lamina propria vessel had no dilatation andinflammatory cell infiltration.

Group B: results of the model control group are shown in B in FIG. 8,wherein a submucosal lamina propria of vagina in the model control grouphad infiltration of a large number of inflammatory cells, disorderedarrangement of loose connective tissues and obvious edema.

Group C: results of the chloroquine-chitosan nanosphere gel group 1,wherein the mass ratio of the chloroquine phosphate to the chitosan was1:3, is shown in C in FIG. 8, wherein connective tissues of a serosallayer of the group were increased, and disordered in arrangement, withobvious edema and infiltration of a small number of inflammatory cells.

Group D: results of the chloroquine-chitosan nanosphere gel group 2,wherein the mass ratio of the chloroquine phosphate to the chitosan was1:0.5, is shown in D in FIG. 8, wherein a submucosal lamina propria ofthe group had obvious edema, and connective tissues of the serosal layerwere increased, and disordered in arrangement, with obvious edema andinflammatory cell infiltration.

Group E: results of the chloroquine phosphate gel group are shown in Ein FIG. 8, wherein local epithelial cells of vaginal mucosa of thechloroquine gel group were deformed and necrotic, and loose connectivetissues in the submucosal lamina propria were slightly disordered, withobvious edema and inflammatory cell infiltration.

(2) Lesion Degree Results of Tested Animals in Histological Examination

TABLE 3 Lesion Degree Statistical Table of Tested Animals inHistological Examination Vagina Disordered loose connective tissues ofThickened lamina serosal Inflammatory Lesion propria and layer cellinfiltration Group degree edema and edema of lamina propria Blank −10/10  10/10  10/10  control ± 0/10 0/10 0/10 group A + 0/10 0/10 0/10++ 0/10 0/10 0/10 +++ 0/10 0/10 0/10 Model − 0/10 2/10 2/10 control ±2/10 3/10 4/10 group B + 5/10 3/10 2/10 ++ 3/10 2/10 2/10 +++ 0/10 0/100/10 Chloroquine- − 4/10 9/10 4/10 chitosan ± 3/10 0/10 3/10nanosphere + 2/10 0/10 3/10 gel group C ++ 1/10 1/10 0/10 +++ 0/10 0/100/10 Chloroquine- − 2/10 8/10 5/10 chitosan ± 2/10 0/10 4/10nanosphere + 3/10 1/10 1/10 gel group D ++ 3/10 0/10 0/10 +++ 0/10 1/100/10 Chloroquine − 1/10 6/10 4/10 phosphate ± 3/10 0/10 4/10 group E +1/10 3/10 2/10 ++ 410 0/10 0/10 +++ 1/10 1/10 0/10 Note: “−” indicatesthat a tissue structure has no obvious abnormality; “±” indicatesextremely mild injury; “+” indicates mild injury; “++” indicatesmoderate injury; and “++” indicates serious injury.

The research results above showed that compared with the chloroquinegel, the chloroquine-chitosan nanosphere gel could obviously improvevaginitis caused by herpes virus. A degree of inflammation of each groupwas compared: the model control group>the chloroquine phosphate groupE>the chloroquine-chitosan nanosphere gel group D>thechloroquine-chitosan nanosphere gel group C>the blank control group.Moreover, the combined application of chloroquine and chitosan couldsignificantly improve vaginal edema, and the results were similar to thedegree of inflammation, which indicated that the combined application ofthe chloroquine and the chitosan had a significant increased therapeuticeffect.

Embodiment 8 Treatment of Condyloma Acuminatum Caused by HPV

1. Experimental Method

A plurality of nude mice (half male and half female) qualified foradaptive observation were selected, HPV-infected wart tissues weredirectly inoculated to the nude mice subcutaneously. A transplantationprocess was strictly in accordance with a principle of asepticoperation. 5 days to 6 days after inoculation of warts, the nude micewith good inoculated parts were selected and randomly and evenly dividedinto 4 groups according to body weight and gender, namely:

Group A: a model control group,

Group B: a chloroquine-chitosan nanosphere gel group 1, wherein a massratio of chloroquine phosphate to chitosan was 1:3,

Group C: a chloroquine-chitosan nanosphere gel group 2, wherein a massratio of chloroquine phosphate to chitosan was 1:0.5, and

Group D: a chloroquine phosphate gel group.

Each group had 6 nude mice (half male and half female). The modelcontrol group was given white vaseline and other groups were all givencorresponding gels. Each group was administrated through skin, and acorresponding administration area on administration parts of animals wasabout 2 cm×2 cm. The medicine was administrated once a day, with 6 hoursfor each contact, and after 6 hours of contact, the medicine wasremoved. The administration was continuously performed for 14 days. Themedicine was administrated 3 times a week for the positive controlgroup, with 6 hours for each contact, and after 6 hours of contact, themedicine was removed. The administration was performed for 14 days. 14days after administration, a wart tissue was taken for fixing andembedding the next day, paraffin wax was continuously cut into sectionsof 4 μm, and after hematoxylin-eosin (HE) staining, a pathologicalcondition of the wart tissue was observed under a microscope.

2. Results

Pathological Examination Results:

(1) Results of warts of Group A showed that the wart tissue could beseen in each animal, an epithelial cell structure of the wart tissue wascomplete, and a koilocytoid cell could be seen locally.

As shown in A in FIG. 9, for the warts, obvious vacuoles existed arounda nucleus of a local epithelial cell, with an obvious nucleolus. (H-Estaining ×200)

(2) Results of warts of Group B showed that the wart tissue could beseen in 6 animals. Epithelial cells of wart tissues of 2 animals (03 and04; 2/6) were degenerated and necrotic locally, and a few koilocytoidcells could be seen; and epithelial cells of wart tissues of 4 animals(01, 02, 05 and 06; 4/6) were complete in structure, and koilocytoidcells could be seen locally.

As shown in B in FIG. 9, for the warts, obvious vacuoles existed arounda nucleus of a local epithelial cell, with an obvious nucleolus. (H-Estaining ×200)

(3) Results of warts of Group C showed that the wart tissue could beseen in 6 animals. Epithelial cells of wart tissues of 3 animals (03, 04and 05; 3/6) were degenerated and necrotic locally, and a fewkoilocytoid cells could be seen; and epithelial cells of wart tissues of3 animals (01, 02 and 06; 3/6) were complete in structure, andkoilocytoid cells could be seen locally.

As shown in C in FIG. 9, for the warts, obvious vacuoles existed arounda nucleus of a local epithelial cell, with an obvious nucleolus. (H-Estaining ×200)

(4) Results of warts of Group D showed that the wart tissue could beseen in 6 animals. Epithelial cells of wart tissues of 4 animals (01,02, 03 and 04; 4/6) were degenerated and necrotic locally, and a fewkoilocytoid cells could be seen; and epithelial cells of wart tissues of2 animals (05 and 06; 2/6) were complete in structure, and koilocytoidcells could be seen locally.

As shown in D in FIG. 9, for the warts, the epidermal cells of the wartshad hyperkeratosis, squamous epithelium could be seen under epidermis,the epithelial cells were degenerated and necrotic locally, and a fewkoilocytoid cells could be seen locally.

The research results above showed that chloroquine had an effect ofclearing viruses of infected cell for the warts caused by HPV, and withthe increase of a chloroquine content, a number of koilocytoid cellsinfected with HPV was decreased. The chloroquine gel group (Group D)could obviously remove infected cells, but could cause localdegeneration and necrosis of tissues. Compared with pure chloroquinegel, the chloroquine-chitosan gel could protect skin while reducing theinfected cells.

Embodiment 9 Killing or Inhibiting Effect on Various Vaginal PathogenicMicroorganisms

In-vitro antibacterial activity detection was performed on thechloroquine-chitosan nanosphere gel group (Group B: chloroquine-chitosannanosphere gel, wherein a mass ratio of chloroquine phosphate tochitosan was 3:1) in the Embodiment 6. 20 g of gel product was addedinto 100 mL of pH 7.0 buffer (negative control was not added with a testsample) to fully dissolve the gel product. 1 mL, 0.5 mL, 0.25 mL and0.125 mL of pH 7.0 buffers added with the test sample were added intocorresponding culture mediums of corresponding bacteria, and cultured ina plate. Pseudomonas aeruginosa, Candida albicans, Escherichia coli andStaphylococcus aureus were resuscitated and diluted, and when abacterial concentration was 100 cfu/mL, each culture medium wasinoculated with an inoculating loop, and observation was started afterculturing for 5 days to 7 days.

In-vitro antibacterial activity detection results showed that thechitosan-chloroquine nanosphere gel with different concentrations couldinhibit growth of the Pseudomonas aeruginosa, the Candida albicans, theEscherichia coli and the Staphylococcus aureus, and the higher theconcentration is, the higher the inhibition rate of bacteria is.Particularly, Candida albicans and Staphylococcus aureus are moresensitive.

The results showed that the chloroquine-chitosan nanosphere gel had agood inhibition effect on various bacteria, but different sensitivities.

According to the present invention, the chloroquine and the chitosanwere jointly applied, which had a synergistic inhibition effect onvarious vaginal pathogenic microorganisms. The medicine of the presentinvention could kill or inhibit various vaginal pathogenicmicroorganisms, such as Staphylococcus aureus (SA), verdigris (EC), betaHemolytic Streptococcus (ST), Candida albicans (CA), Ureaplasmaurealyticum (Uu), Trichomonas vaginalis and Bacteroides fragilis, andherpes simplex virus type 2 (HSV-2). In addition, the medicine of thepresent invention could inhibit adhesion of a pathogen and a vaginalepithelial cell of a host, and prevent and treat various vaginitis, suchas Candida albicans vaginitis, trichomonal vaginitis, bacterialvaginosis, genital herpes virus and other external genitalia infections.

Embodiment 10 Preliminary Clinical Research Results

It was found from application of the chloroquine-chitosan nanosphere ofthe present invention to 10 women suffering from vaginitis caused bycandida infection that no patient reported a side effect and an adversereaction in a treatment process, and no side effect and adverse reactionwere found after 6 months of follow-up, and a total effective rate ofthe chloroquine-chitosan nanosphere treatment group was obviouslyincreased by 30% compared with that of the chloroquine treatment groupalone.

The present invention was applied to treatment of warts of 30 patientscaused by HPV infection, and after 2 weeks to 3 weeks of treatment, thewarts of the 30 patients were found to naturally fall off without anyadverse reactions.

The chloroquine nanosphere of the present invention has good therapeuticeffects on external genitalia infection including viral vaginitis,condyloma acuminatum, bacterial vaginosis, fungal-infectious vaginitisor trichomonal vaginitis, and other cutaneous warts including flat wartsand warts at non-genital parts caused by HPV infection.

Obviously, the above-mentioned embodiments of the invention are merelyexamples for clearly illustrating the invention, but are not intended tolimit the implementations of the invention. For those of ordinary skillsin the art, other different forms of changes or variations can be madeon the basis of the above description. It is not necessary or possibleto exhaust all the implementations here. Any change, equivalentsubstitution, and improvement made within the spirit and principle ofthe invention shall fall within the scope of protection of the claims ofthe invention.

1. A chloroquine nanosphere, wherein the chloroquine nanospherecomprises a water-soluble nanosphere carrier, and chloroquine or achloroquine derivative a mass ratio of the chloroquine or thechloroquine derivative to the water-soluble nanosphere carrier duringpreparation ranges from 1:3 to 1:5: a loading rate of the chloroquine orthe chloroquine derivative in the prepared chloroquine nanosphere rangesfrom 3.0% to 21.6%: the water-soluble nanosphere carrier iswater-soluble chitosan; a deacetylation degree of the water-solublechitosan ranges from 80% to 95%, and a viscosity-average molecularweight thereof ranges from 3000 to 5000; and the chloroquine derivativeis selected from one or more of hydroxychloroquine, chloroquinephosphate or chloroquine sulfate.
 2. The chloroquine nanosphereaccording to claim 1, wherein a preparation method thereof comprises thefollowing steps: S1: after dissolving the water-soluble nanospherecarrier, adding the chloroquine or the chloroquine derivative, andevenly mixing and stirring to form an aqueous phase; S2: adding anemulsifier into an oil phase matrix to form an oil phase; adding theaqueous phase into the oil phase, wherein a volume ratio of the aqueousphase to the oil phase is 1:1 to 1:6; and emulsifying at 10000-20000r/min for 10-30 minutes to obtain a nanosphere emulsion; and S3: addinga precipitant into the nanosphere emulsion, after evenly mixing,standing for 2-8 minutes, then dehydrating, performing high-speedcentrifugation and cleaning, and drying to obtain the chloroquinenanosphere; wherein conditions for the high-speed centrifugation is tocentrifuge at 1500-2000 r/min for 5-10 minutes; the oil phase matrix isselected from one or more of corn oil, olive oil, peanut oil, soybeanoil, or rapeseed oil; the emulsifier is selected from one or more ofTween-20, Tween-80 or Span 80; the precipitant is a sodiumhydroxide-n-propanol mixed solution. 3-6. (canceled)
 7. The chloroquinenanosphere according to claim 2, wherein a volume ratio of theemulsifier to the precipitant is (1-1.5):(30-50).
 8. (canceled)
 9. Thechloroquine nanosphere according to claim 2, wherein a pH value of theprecipitant ranges from 8.5 to 10.0.
 10. Application of the chloroquinenanosphere according to claim 1 in preparing an antibacterial and/orantiviral product, wherein the chloroquine nanosphere is prepared intogel for use.
 11. Application of the chloroquine nanosphere according toclaim 1 in preparing a product for preventing and treating externalgenitalia infection and/or flat warts.
 12. The application according toclaim 11, wherein the external genitalia infection comprises viralvaginitis, condyloma acuminatum, bacterial vaginosis, fungal-infectiousvaginitis or trichomonal vaginitis.
 13. (canceled)
 14. A product forpreventing and treating external genitalia infection and/or flat warts,wherein the product comprises the chloroquine nanosphere according toclaim
 1. 15. (canceled)
 16. The product according to claim 14, whereinthe chloroquine nanosphere accounts for 1% to 6% of a total weight ofthe product.
 17. The product according to claim 14, wherein a dosageform of the product is gel.
 18. Application of the chloroquinenanosphere according to claim 2 in preparing an antibacterial and/orantiviral product, wherein the chloroquine nanosphere is prepared intogel for use.
 19. Application of the chloroquine nanosphere according toclaim 2 in preparing a product for preventing and treating externalgenitalia infection and/or other cutaneous flat warts.
 20. A product forpreventing and treating external genitalia infection and/or flat warts,characterized in that, the product comprises the chloroquine nanosphereaccording to claim
 2. 21. A product for preventing and treating externalgenitalia infection and/or flat warts, characterized in that, theproduct comprises the chloroquine nanosphere according to claim
 7. 22. Aproduct for preventing and treating external genitalia infection and/orflat warts, characterized in that, the product comprises the chloroquinenanosphere according to claim
 9. 23. The product according to claim 16,characterized in that, a dosage form of the product is gel.